For many diagnostic and/or treatment purposes, it is highly desirable to be able to measure the number of infectious viral particles in a sample or preparation. For example, rapid and accurate measurement of viral growth is necessary for treatment of viral infections and for monitoring whether a therapeutic strategy is effective in inhibiting viral growth, or whether the virus has developed resistance to compounds used for treatment. Also, as viral vectors are primary delivery vehicles for gene therapy, it is critical that the quantity of the viral particles or dosage in a clinical preparation be rapidly and accurately determined.
Although total particle measurement can be made by techniques such as electron microscopy of viral preparations or measurement of total nucleic acid content, the current “gold standard” for measurement of viral infectivity is the plaque assay. The conventional plaque assay is performed by applying a dilute solution of viruses to a monolayer of susceptible host cells, allowing virus particles to adsorb to cells and then overlaying the cells with a semi-solid agar. Isolated infected cells then produce virus progeny that spread to and infect neighboring cells. Several cycles of virus growth and spread eventually produce a “plaque,” a macroscopic island of dying or dead cells surrounded by a sea of uninfected cells. If the initial viral solution is sufficiently dilute, each infectious virus particle in the solution will initiate an infection and cause the formation of one plaque. The total number of plaques thus determines the initial number of infectious particles within the sample. Manual counting of plaques provides the sample infectivity, expressed as a number of plaque forming units (PFU) per unit volume. The size of the plaques also provides a measure of virus infectivity, reflecting the rate and productivity of the virus infections.
This classical plaque assay, however, suffers from the disadvantages that it is time consuming and lacks sufficient sensitivity, in particular because the virus particles released from an infected cell is limited in its ability to spread and reach additional host cells to initiate further rounds of infections.
The instant invention addresses the need for a more accurate method of quantifying infectious viral particles in a population.
If the classical plaque assay is performed with a fluid overlay, instead of an agar overlay, then larger regions of cell death form. These larger regions often have the appearances of comets, as such this modified plaque assay is known as “comet assays.” The formation of comets was first reported 35 years ago in studies of vaccinia virus (VV) spread (Appleyard et al., An antigenic difference between intracellular and extracellular rabbitpox virus. J Gen Virol 13, 9-17 (1971)), and comet assays have been most widely used in VV research (Payne, Significance of extracellular enveloped virus in the in vitro and in vivo dissemination of vaccinia. J Gen Virol 50, 89-100 (1980), Blasco et al., Dissociation of progeny vaccinia virus from the cell membrane is regulated by a viral envelope glycoprotein: effect of a point mutation in the lectin homology domain of the A34R gene. J Virol 67, 3319-25 (1993), Katz et al., Identification of second-site mutations that enhance release and spread of vaccinia virus. J. Virol 76, 11637-44 (2002); Law et al., Antibody-sensitive and antibody-resistant cell-to-cell spread by vaccinia virus: role of the A33R protein in antibody-resistant spread. J Gen Virol 83, 209-22 (2002); Mathew et al., The extracellular domain of vaccinia virus protein B5R affects plaque phenotype, extracellular enveloped virus release, and intracellular actin tail formation. J Virol 72, 2429-38 (1998). Vanderplasschen et al., Antibodies against vaccinia virus do not neutralize extracellular enveloped virus but prevent virus release from infected cells and comet formation. J Gen Virol 78 (Pt 8), 2041-8 (1997); and Wyatt et al., Highly attenuated smallpox vaccine protects mice with and without immune deficiencies against pathogenic vaccinia virus challenge. Proc Natl Acad Sci USA 101, 4590-5 (2004)), and to a more limited extent for herpes simplex virus (Shinkai, Plaque morphology of herpes simplex virus in various cells under liquid overlay as a marker for its type differentiation. Jpn J Microbiol 19, 459-62 (1975)), variola virus (Reeves et al., Disabling poxvirus pathogenesis by inhibition of Abl-family tyrosine kinases. Nat Med 11, 731-9 (2005)), and influenza virus (Gambaryan et al., Differences in the biological phenotype of low-yielding (L) and high-yielding (H) variants of swine influenza virus A/NJ/11/76 are associated with their different receptor-binding activity. Virology 247, 223-31 (1998); Matrosovich et al., Overexpression of the alpha-2,6-sialyltransferase in MDCK cells increases influenza virus sensitivity to neuraminidase inhibitors. J Virol 77, 8418-25 (2003)).